In recent years, highly pathogenic avian influenza A virus strains with H5N1 subtype have become entrenched in poultry worldwide and pose a growing threat to human health. Because of continuous variation of this virus, developing anew anti-influenza drug has become an urgent and major task for all countries. Demonstration of three-dimensional structures of the proteins which are related to the influenza virus has important scientific significance for understanding the viral replication, and is highly valuable for the development of anti-influenza viral drugs.
The RNA genome of the influenza virus contains 8 RNA segments which encode 11 virus-specific proteins. Influenza virus RNA-dependent RNA polymerase is a heterotrimeric complex (PA, PB1 and PB2) harboring several enzymatic activities for catalyzing both viral RNA replication and transcription, and acts to maintain virus life cycle. In particular, the high conservation and low mutation ratio of subunits PA, PB1 and PB2, enabled it as a viable target to design the anti-influenza drugs.
In recent years, it has been known that PB1 (SEQ ID NO: 2) subunit alone can catalyze viral RNA replication and transcription; PB2 subunit binds the 5′ cap of host pre-mRNAs, which are subsequently cleaved by the viral endonuclease, hitherto thought to reside in the PB2 or PB1 (SEQ ID NO: 2) subunits.
Compared to the other two subunits, the mechanism of the PA subunit remained elusive. PA is an important protein in the polymerase heterotrimer and may be required for replication and transcription of viral RNA (vRNA) and endonuclease cleavage of the cap RNA primer. It reportedly induces proteolysis of the viral and host proteins and may also be involved in virus assembly. However, the molecular mechanism of PA remains unclear. Hereby, investigation of the PA structure is significantly important to study the whole RNA polymerase complex.
Analysis of protein structures is a very useful tool to understand protein function. Especially important to the study of the function of the complex is exploration of the whole complex. However, due to various difficulties, the structure of this protein complex has not been resolved.
In Chinese patents No. CN 200810100840.X and CN 200810083994.2 submitted on Feb. 22 and May 2 in 2008, present inventors disclosed three dimensional crystal structure of influenza A virus PA (PAC, residues 257-716 of SEQ ID NO: 1) in complex with the PA-binding region of PB1 (PB1N, residues 1-25 of SEQ ID NO: 2). Present inventors published the structure of avian H5N1 influenza A virus PA (PAC, residues 257-716 of SEQ ID NO: 1) in complex with the PA-binding region of PB1 (PB1N, residues 1-25 of SEQ ID NO:2) (He X et al. Nature, August 2008, 454(7208):1123-6).
In order to obtain a completely three-dimensional crystal structure of the polymerase complex which consists of PA, PB1, and PB2 subunits, present inventors have conducted the following research.